Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

Photochemical activation of drugs for the treatment of therapy-resistant cancers.

Resistance to chemotherapy, molecular targeted therapy as well as radiation therapy is a major obstacle for cancer treatment. Cancer resistance may be exerted through multiple different mechanisms which may be orchestrated as observed in multidrug resistance (MDR). Cancer resistance may be intrinsic or acquired and often leaves patients without any treatment options. Strategies for alternative treatment modalities for resistant cancer are therefore highly warranted. Photochemical internalization (PCI) is a technology for cytosolic delivery of macromolecular therapeutics based on the principles of photodynamic therapy (PDT). The present report reviews the current knowledge of PCI of therapy-resistant cancers. In summary, PCI may be able to circumvent several of the major mechanisms associated with resistance towards chemotherapeutics including increased expression of drug efflux pumps, altered intracellular drug distribution and increased ROS scavenging. Current data also suggest PCI of targeted toxins as highly effective in cancers resistant to clinically available targeted therapy such as monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). PCI may therefore, in general, represent a future treatment option for cancers resistant to other therapies.

Sustained ERK [corrected] inhibition by EGFR targeting therapies is a predictive factor for synergistic cytotoxicity with PDT as neoadjuvant therapy.

BACKGROUND: Tyrosin kinase inhibitors (TKIs) and monoclonal antibodies aimed to target epidermal growth factor receptor (EGFR) have shown limited effect as monotherapies and drug resistance is a major limitation for therapeutic success. Adjuvant therapies to EGFR targeting therapeutics are therefore of high clinical relevance. METHODS: Three EGFR targeting drugs, Cetuximab, Erlotinib and Tyrphostin AG1478 were used in combination with photodynamic therapy (PDT) in two EGFR positive cell lines, A-431 epidermoid skin carcinoma and WiDr colorectal adenocarcinoma cells. The amphiphilic meso-tetraphenylporphine with 2 sulphonate groups on adjacent phenyl rings (TPPS(2a)) was utilized as a photosensitizer for PDT. The cytotoxic outcome of the combined treatments was evaluated by cell counting and MTT. Cellular signalling was explored by Western blotting. RESULTS: PDT as neoadjuvant to Tyrphostin in A-431 cells as well as to Tyrphostin or Erlotinib in WiDr cells revealed synergistic cytotoxicity. In contrast, Erlotinib or Cetuximab combined with neoadjuvant PDT induced an antagonistic effect on cell survival of A-431 cells. Neoadjuvant PDT and EGFR targeting therapies induced a synergistic inhibition of ERK as well as synergistic cytotoxicity only when the EGFR targeting monotherapies caused a prolonged ERK inhibition. There were no correlation between EGFR inhibition by the EGFR targeting monotherapies or the combined therapies and the cytotoxic outcome combination-therapies. CONCLUSIONS: The results suggest that sustained ERK inhibition by EGFR targeting monotherapies is a predictive factor for synergistic cytotoxicity when combined with neoadjuvant PDT. GENERAL SIGNIFICANCE: The present study provides a rationale for selecting anticancer drugs which may benefit from PDT as adjuvant therapy.

Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization.

We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.

Photochemical internalization (PCI) of HER2-targeted toxins: synergy is dependent on the treatment sequence.

BACKGROUND: Photochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen ((1)O(2)). METHODS: The human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab-saporin, was evaluated in combination with PCI using TPCS(2a) (Amphinex®), a new photosensitizer approved for clinical use. RESULTS: PCI synergistically enhanced the cytotoxicity of trastuzumab-saporin on trastuzumab-resistant HER2(+) Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment ("light after" strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS(2a)-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by (1)O(2) generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane. CONCLUSIONS: Administration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins. GENERAL SIGNIFICANCE: PCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the "light first" procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.

Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

Background: Immune-checkpoint inhibitors (ICIs) represent a revolution in cancer therapy and are currently implemented as standard therapy within several cancer indications. Nevertheless, the treatment is only effective in a subset of patients, and immune-related adverse effects complicate the improved survival. Adjuvant treatments that can improve the efficacy of ICIs are highly warranted, not only to increase the response rate, but also to reduce the therapeutic ICI dosage. Several treatment modalities have been suggested as ICI adjuvants including vascular targeted treatments and photodynamic therapy (PDT). Photochemical internalization (PCI) is a drug delivery system, based on PDT. PCI is long known to generate an immune response in murine models and was recently shown to enhance the cellular immune response of a vaccine in a clinical study. In the present work we evaluated PCI in combination with the vascular targeting toxin VEGF121/rGel with respect to induction of immune-mediated cell death as well as in vitro ICI enhancement. Methods: DAMP signaling post VEGF121/rGel-PCI was assessed in CT26 and MC38 murine colon cancer cell lines. Hypericin-PDT, previously indicated as an highly efficient DAMP inducer (but difficult to utilize clinically), was used as a control. ATP release was detected by a bioluminescent kit while HMGB1 and HSP90 relocalization and secretion was detected by fluorescence microscopy and western blotting. VEGF121/rGel-PCI was further investigated as an αCTLA enhancer in CT26 and MC38 tumors by measurement of tumor growth delay. CD8+ Dependent efficacy was evaluated in vivo using a CD8+ antibody. Results: VEGF121/rGel-PCI was shown to induce increased DAMP signaling as compared to PDT and VEGF121/rGel alone and the magnitude was found similar to that induced by Hypericin-PDT. Furthermore, a significant CD8+ dependent enhanced αCTLA-4 treatment effect was observed when VEGF121/rGel-PCI was used as an adjuvant in both tumor models. Conclusions: VEGF121/rGel-PCI describes a novel concept for ICI enhancement which induces a rapid CD8+ dependent tumor eradication in both CT26 and MC38 tumors. The concept is based on the combination of intracellular ROS generation and vascular targeting using a plant derived toxin and will be developed towards clinical utilization.

Corrigendum: Light-enhanced VEGF121/rGel induce immunogenic cell death and increase the antitumor activity of αCTLA4 treatment.

[This corrects the article DOI: 10.3389/fimmu.2023.1278000.].

Photochemical internalization of tumor-targeted protein toxins.

Photochemical internalization (PCI) is a method for intracellular delivery of hydrophilic macromolecular drugs with intracellular targets as well as other drugs with limited ability to penetrate cellular membranes. Such drugs enter cells by means of endocytosis and are to a large extent degraded by hydrolytic enzymes in the lysosomes unless they possess a mechanism for cytosolic translocation. PCI is based on photodynamic therapy (PDT) specifically targeting the endosomes and lysosomes of the cells, so that the drugs in these vesicles can escape into the cytosol from where they can reach their targets. The preferential retention of the photosensitizer (PS) in tumor tissue in combination with controlled light delivery makes PCI relatively selective for cancer tissue. The tumor specificity of PCI can be further increased by delivery of drugs that selectively target the tumors. Indeed, this has been shown by PCI delivery of several targeted protein toxins. Targeted protein toxins may be regarded as ideal drugs for PCI delivery, and may represent the clinical future for the PCI technology.

Inhibiting autophagy increases the efficacy of low-dose photodynamic therapy.

Rupture and permeabilization of endocytic vesicles can be triggered by various causes, such as pathogenic invasions, amyloid proteins, and silica crystals leading to cell death and degeneration. A cellular quality control process, called lysophagy was recently described to target damaged lysosomes for autophagic sequestration within isolation membranes in order to protect the cell from the consequences of lysosomal leakage. This protective process, however, might interfere with treatment conditions, such as photodynamic therapy (PDT) and the intracellular drug delivery method photochemical internalization (PCI). PCI-induced permeabilization of endosomes and lysosomes is purposely triggered to release drugs that are sequestered in these organelles into the cytosol in order to synergistically kill cancer cells. Here, we show that photochemical treatment with the PCI-photosensitizer TPCS2a/fimaporfin results in both induction of autophagy and inhibition of the autophagic flux. The autophagic response is accompanied by recruitment of ubiquitin (Ubq), p62, and microtubule-associated protein 1A/1B-light chain 3 (LC3) to damaged vesicles, marked by Galectin 3 (Gal3). Furthermore, ultrastructural analysis revealed a homogenously thick p62-positive layer surrounding these permeabilized vesicles. Although p62 seems to be important during the selective autophagic sequestration, we show that its presence is not essential for the effective removal of damaged vesicles or the recovery of the lysosomal content. An active autophagic response and the presence of p62, however, is important for cancer cells to survive low-dose TPCS2a-PDT. Thus, targeting both p62 and autophagy together and independently, in a light-controlled/PCI based delivery of cancer therapeutics could increase the effectiveness of the treatment regime.

RAB5A expression is a predictive biomarker for trastuzumab emtansine in breast cancer.

HER2 is a predictive biomarker for HER2-targeted therapeutics. For antibody-drug conjugates (ADCs; e.g., trastuzumab emtansine (T-DM1)), HER2 is utilized as a transport gate for cytotoxic agents into the cell. ADC biomarkers may therefore be more complex, also reflecting the intracellular drug transport. Here we report on a positive correlation between the early endosome marker RAB5A and T-DM1 sensitivity in five HER2-positive cell lines. Correlation between RAB5A expression and T-DM1 sensitivity is confirmed in breast cancer patients treated with trastuzumab emtansine/pertuzumab in the I-SPY2 trial (NCT01042379), but not in the trastuzumab/paclitaxel control arm. The clinical correlation is further verified in patients from the KAMILLA trial (NCT01702571). In conclusion, our results suggest RAB5A as a predictive biomarker for T-DM1 response and outline proteins involved in endocytic trafficking as predictive biomarkers for ADCs.

Photochemical internalization: a new tool for gene and oligonucleotide delivery.

Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles. Upon activation by light such photosensitizers induce a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to increase the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, plasmids, adenovirus, various oligonucleotides, dendrimer-based delivery of chemotherapeutica and unconjugated chemotherapeutica such as bleomycin and doxorubicin. This review will present the basis for the PCI concept and the most recent significant developments.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.

Photochemically-Induced Release of Lysosomal Sequestered Sunitinib: Obstacles for Therapeutic Efficacy.

Lysosomal accumulation of sunitinib has been suggested as an underlying mechanism of resistance. Here, we investigated if photochemical internalization (PCI), a technology for cytosolic release of drugs entrapped in endosomes and lysosomes, would activate lysosomal sequestered sunitinib. By super-resolution fluorescence microscopy, sunitinib was found to accumulate in the membrane of endo/lysosomal compartments together with the photosensitizer disulfonated tetraphenylchlorin (TPCS2a). Furthermore, the treatment effect was potentiated by PCI in the human HT-29 and the mouse CT26.WT colon cancer cell lines. The cytotoxic outcome of sunitinib-PCI was, however, highly dependent on the treatment protocol. Thus, neoadjuvant PCI inhibited lysosomal accumulation of sunitinib. PCI also inhibited lysosomal sequestering of sunitinib in HT29/SR cells with acquired sunitinib resistance, but did not reverse the resistance. The mechanism of acquired sunitinib resistance in HT29/SR cells was therefore not related to lysosomal sequestering. Sunitinib-PCI was further evaluated on HT-29 xenografts in athymic mice, but was found to induce only a minor effect on tumor growth delay. In immunocompetent mice sunitinib-PCI enhanced areas of treatment-induced necrosis compared to the monotherapy groups. However, the tumor growth was not delayed, and decreased infiltration of CD3-positive T cells was indicated as a possible mechanism behind the failed overall response.

Photodynamic therapy with an endocytically located photosensitizer cause a rapid activation of the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2 terminal kinase with opposing effects on cell survival.

Photochemical internalization (PCI) is a method for release of endosomally/lysosomally trapped drugs into the cell cytosol. PCI is based on photosensitizers that accumulate in the membranes of endosomes and lysosomes. Light exposure generates reactive oxygen species that cause membrane rupture and subsequently drug release. PCI can be considered as a combination therapy of photodynamic therapy (PDT) and the administrated drug. The present work reports on mitogen-activated protein kinase signaling after PDT with the endocytically located photosensitizer TPPS(2a) (meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings) as used for PCI in two cancer cell lines: NuTu-19 and WiDr. Both extracellular signal-regulated kinase (ERK) and p38 were activated immediately after PDT. The photochemically induced ERK phosphorylation was enhanced by epidermal growth factor stimulation to a level above that obtainable with epidermal growth factor alone. Expression of the ERK phosphatase, MAPK phosphatase-1, was increased 2 h after PDT but was not the cause of ERK dephosphorylation observed simultaneously. A transient activation of c-Jun NH2 terminal kinase was also observed after PDT but only in the NuTu-19 cells. Using suitable inhibitors, it is shown here that the p38 signal is a death signal, whereas c-Jun NH2 terminal kinase rescues cells after PDT. No direct connection was observed between PDT-induced ERK activation and toxicity of the treatment. The present results document the importance of the mitogen-activated protein kinases in TPPS(2a)-PDT-induced cytotoxicity.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Light-enhanced VEGF121/rGel: A tumor targeted modality with vascular and immune-mediated efficacy.

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.

Y1068 phosphorylation is the most sensitive target of disulfonated tetraphenylporphyrin-based photodynamic therapy on epidermal growth factor receptor.

Photodynamic therapy (PDT) is an anticancer therapy that utilizes the cytotoxic properties of a photosensitizer (PS) when combined with exposure to light. Photochemical internalization (PCI) is a drug delivery method for macromolecules based on PDT with endo-lysosomal localizing PSs, and synergistic effects can be obtained by PCI of EGFR targeting drugs. In this report the effects of PDT with two endo-lysosomal localizing PSs on EGFR are described. The experiments were performed in EGFR-positive cell-lines in vitro and also in a subcutaneous tumour-model in mice. In PCI, the PSs are transported from the plasma membrane to endocytic vesicles by endocytosis and some of the PS can therefore be retained at the plasma membrane. Two distinct treatment conditions with different amounts of the PS on the plasma membrane were therefore studied in vitro. The expression of total and phosphorylated EGFR was analyzed on Western blots and EGF-binding to EGFR was evaluated by fluorescence microscopy of Alexa488-labelled EGF. The results showed that PDT, as utilized in PCI, caused inhibition of EGF-stimulated EGFR phosphorylation on Y1068 in NuTu-19 cells, but not in WiDr cells. PDT performed with more PS on the plasma membrane of NuTu-19 cells caused in addition inhibition of EGF binding and also lack of recognition by antibodies towards sequences in the intracellular domain of EGFR. In vivo, total EGFR was reduced 24h after PDT in WiDr tumours. This report indicates EGF-stimulated phosphorylation on Y1068 as the most sensitive target on EGFR to PDT with amphiphilic PSs.

Photochemical delivery of bleomycin induces T-cell activation of importance for curative effect and systemic anti-tumor immunity.

Photochemical internalization (PCI) is a technology to enhance intracellular drug delivery by light-induced translocation of endocytosed therapeutics into the cytosol. The aim of this study was to explore the efficacy of PCI-based delivery of bleomycin and the impact on systemic anti-tumor immunity. Mouse colon carcinoma cells (CT26.CL25), stably expressing the bacterial ß-galactosidase, were inoculated into the legs of athymic or immuno-competent BALB/c mice strains. The mice were injected with the photosensitizer AlPcS2a and bleomycin (BLM) prior to tumor light exposure from a 670nm diode laser. Photochemical activation of BLM was found to induce synergistic inhibition of tumor growth as compared to the sum of the individual treatments. However, a curative effect was not observed in the athymic mice exposed to 30J/cm2 of light while >90% of the thymic mice were cured after exposure to only 15J/cm2 light. Cured thymic mice, re-challenged with CT26.CL25 tumor cells on the contralateral leg, rejected 57-100% of the tumor cells inoculated immediately and up to 2months after the photochemical treatment. T-cells from the spleen of PCI-treated mice were found to inhibit the growth of CT26.CL25 cells in naïve thymic mice with a 60% rejection rate. The results show that treatment of CT26.CL25 tumors in thymic mice by PCI of BLM induces a systemic anti-tumor immunity.

Development of resistance to photodynamic therapy (PDT) in human breast cancer cells is photosensitizer-dependent: Possible mechanisms and approaches for overcoming PDT-resistance.

Here we report on the induction of resistance to photodynamic therapy (PDT) in the ABCG2-high human breast cancer cell line MA11 after repetitive PDT, using either Pheophorbide A (PhA) or di-sulphonated meso-tetraphenylchlorin (TPCS2a) as photosensitizer. Resistance to PhA-PDT was associated with enhanced expression of the efflux pump ABCG2. TPCS2a-PDT-resistance was neither found to correspond with lower TPCS2a-accumulation nor reduced generation of reactive oxygen species (ROS). Cross-resistance to chemotherapy (doxorubicin) or radiotherapy was not observed. TPCS2a-PDT-resistant cells acquired a higher proliferation capacity and an enhanced expression of EGFR and ERK1/2. p38 MAPK was found to be a death-signalling pathway in the MA11 cells post TPCS2a-PDT, contrasting the MA11/TR cells in which PDT generated a sustained phosphorylation of p38 that had lost its death-mediated signalling, and an abrogated activation of its downstream effector MAPKAPK2. No difference in apoptosis, necrosis or autophagy responses was found between the treated cell lines. Development of TPCS2a-PDT resistance in the MDA-MB-231 cell line was also established, however, p38 MAPK did not play a role in the PDT-resistance. MCF-7 cells did not develop TPCS2a-PDT-resistance. Photochemical internalisation (PCI) of 1 pM of EGF-saporin induced equal strong cytotoxicity in both MA11 and MA11/TR cells. In conclusion, loss of p38 MAPK-inducing death signalling is the main mechanism of resistance to TPCS2a-PDT in the MA11/TR cell line. This work provides mechanistic knowledge of intrinsic and acquired PDT-resistance which is dependent on choice of photosensitizer, and suggests PCI as a rational therapeutic intervention for the elimination of PDT-resistant cells.